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Tetracosahexaenoic acid (24:6ω‐3) is an intermediate in the conversion of 18:3ω‐3 to 22:6ω‐3 in mammals. There is limited information about whether cells can assimilate and metabolize exogenous 24:6ω‐3. This study compared the effect of incubation with 24:6ω‐3 on the fatty acid composition of two related cell types, primary CD3+ T lymphocytes and Jurkat T cell leukemia, which differ in the integrity of the polyunsaturated fatty acid (PUFA) biosynthesis pathway. 24:6ω‐3 was only detected in either cell type when cells were incubated with 24:6ω‐3. Incubation with 24:6ω‐3 induced similar increments in the amount of 22:6ω‐3 in both cell types and modified the homeoviscous adaptations fatty acid composition induced by activation of T lymphocytes. The effect of incubation with 18:3ω‐3 compared to 24:6ω‐3 on the increment in 22:6ω‐3 was tested in Jurkat cells because primary T cells cannot convert 18:3ω‐3 to 22:6ω‐3. The increment in the 22:6ω‐3 content of Jurkat cells incubated with 24:6ω‐3 was 19.5‐fold greater than that of cells incubated with 18:3ω‐3. Acyl‐coA oxidase siRNA knockdown decreased the amount of 22:6ω‐3 and increased the amount of 24:6ω‐3 in Jurkat cells. These findings show exogenous 24:6ω‐3 can be incorporated into primary human T lymphocytes and Jurkat cells and induces changes in fatty acid composition consistent with its conversion to 22:6ω‐3 via a mechanism involving peroxisomal β‐oxidation that is regulated independently from the integrity of the upstream PUFA synthesis pathway. One further implication is that consuming 24:6ω‐3 may be an effective alternative means of achieving health benefits attributed to 20:5ω‐3 and 22:6ω‐3. [ABSTRACT FROM AUTHOR] more...

IntroductionComorbidity with metabolic diseases indicates that lipid metabolism plays a role in the etiology of Alzheimer's disease (AD). Comprehensive lipidomic analysis can provide new insights into the altered lipid metabolism in AD.MethodIn this study, a total 349 serum lipids were measured in 806 participants enrolled in the Alzheimer's Disease Neuroimaging Initiative Phase 1 cohort and analyzed using lipid-set enrichment statistics, a data mining method to find coregulated lipid sets.ResultsWe found that sets of blood lipids were associated with current AD biomarkers and with AD clinical symptoms. AD diagnosis was associated with 7 of 28 lipid sets of which four also correlated with cognitive decline, including polyunsaturated fatty acids. Cerebrospinal fluid amyloid beta (Aβ1-42) correlated with glucosylceramides, lysophosphatidylcholines and unsaturated triacylglycerides; cerebrospinal fluid total tau and brain atrophy correlated with monounsaturated sphingomyelins and ceramides, in addition to EPA-containing lipids.DiscussionAD-associated lipid sets indicated that lipid desaturation, elongation, and acyl chain remodeling processes are disturbed in AD subjects. Monounsaturated lipid metabolism was important in early stages of AD, whereas the polyunsaturated lipid metabolism was associated with later stages of AD. Our study provides several new hypotheses for studying the role of lipid metabolism in AD. more...

The enzymatic pathway of cholesterol biosynthesis has been well characterized. However, there remain several potential interacting proteins that may play ancillary roles in the regulation of cholesterol production. Here, we identified ERG28 (chromosome 14 open reading frame 1 [C14orf1]), a homologue of the yeast protein Erg28p, as a player in mammalian cholesterol synthesis. ERG28 is conserved from yeast to humans but has been largely overlooked in mammals. Using quantitative RT-PCR, luciferase assays, and publicly available chromatin immunoprecipitation sequencing data, we found that transcription of this gene is driven by the transcription factor SREBP-2, akin to most cholesterol synthesis enzymes, as well as identifying sterol-responsive elements and cofactor binding sites in its proximal promoter. Based on a split luciferase system, ERG28 interacted with itself and two enzymes of cholesterol synthesis (NSDHL and SC4MOL). Huh7 ERG28-KO cell lines were generated, revealing reduced total cholesterol levels in sterol-depleted environments. In addition, radiolabeled metabolic flux assays showed a 60–75% reduction in the rate of cholesterol synthesis in the KO versus wild-type cells, which could be rescued by expression of ectopic ERG28. Unexpectedly, KO of ERG28 also impaired the activation of SREBP-2 under sterol-replete conditions, by a yet-to-be defined mechanism. These results indicate that ERG28 is clearly involved in cholesterol synthesis, although the precise role this noncatalytic protein plays in this complex metabolic pathway remains to be fully elucidated. A deeper understanding of ERG28, and other ancillary proteins of cholesterol synthesis, may help inform therapeutic strategies for diseases associated with aberrant cholesterol metabolism. more...

Nanodiscs are discoidal lipoproteins that have often been used as vehicles to study membrane proteins in their native configuration. Nanodiscs have been primarily made from synthetic lipids. However, nanodiscs also offer a format by which native lipids can be studied in their natural configuration. Here, we present a method to synthesize nanodiscs from bacterial total lipid extracts using the biothreat agent, Yersinia pestis, as a proof-of-concept. The creation of nanoparticles entirely composed of bacterial lipids supports membrane characterization and vaccine antigen discovery without the inherent safety concerns associated with live bacterial cells of this Tier 1 select agent pathogen. [Display omitted] • Protocol developed for assembly of nanodiscs (NDs) from total bacterial lipid extracts. • Efficiency of heterogenous ND assembly depends on lipid to protein mass ratio and temperature. • Post-assembly centrifugation step removes large particles similar to size exclusion chromatography. [ABSTRACT FROM AUTHOR] more...

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina. more...

Gram-negative bacteria possess an asymmetric outer membrane (OM) composed primarily of lipopolysaccharides (LPSs) on the outer leaflet and phospholipids (PLs) on the inner leaflet. The loss of this asymmetry due to mutations in the LPS biosynthesis or transport pathways causes the externalization of PLs to the outer leaflet of the OM and leads to OM permeability defects. Here, we used metabolic labeling to detect a compromised OM in intact bacteria. Phosphatidylcholine synthase expression in Escherichia coli allowed for the incorporation of exogenous propargylcholine into phosphatidyl(propargyl)choline and exogenous 1-azidoethyl-choline (AECho) into phosphatidyl(azidoethyl)choline (AEPC), as confirmed by LC/MS analyses. A fluorescent copper-free click reagent poorly labeled AEPC in intact wild-type cells but readily labeled AEPC from lysed cells. Fluorescence microscopy and flow cytometry analyses confirmed the absence of significant AEPC labeling from intact wild-type E. coli strains and revealed significant AEPC labeling in an E. coli LPS transport mutant (lptD4213) and an LPS biosynthesis mutant (E. coli lpxC101). Our results suggest that metabolic PL labeling with AECho is a promising tool for detecting a compromised bacterial OM, revealing aberrant PL externalization, and identifying or characterizing novel cell-active inhibitors of LPS biosynthesis or transport.­ more...

Apolipoprotein F (ApoF) modulates lipoprotein metabolism by selectively inhibiting cholesteryl ester transfer protein activity on LDL. This ApoF activity requires that it is bound to LDL. How hyperlipidemia alters total plasma ApoF and its binding to LDL are poorly understood. In this study, total plasma ApoF and LDL-bound ApoF were quantified by ELISA (n = 200). Plasma ApoF was increased 31% in hypercholesterolemic plasma but decreased 20% in hypertriglyceridemia. However, in donors with combined hypercholesterolemia and hypertriglyceridemia, the elevated triglyceride ameliorated the rise in ApoF caused by hypercholesterolemia alone. Compared with normolipidemic LDL, hypercholesterolemic LDL contained ∼2-fold more ApoF per LDL particle, whereas ApoF bound to LDL in hypertriglyceridemia plasma was more...

In light of the importance of epoxyeicosatrienoic acids (EETs) in mammalian pathophysiology, a nonenzymatic route that might form these monoepoxides in cells is of significant interest. In the late 1970s, a simple system of arranging linoleic acid molecules on a monolayer on silica was devised and shown to yield monoepoxides as the main autoxidation products. Here, we investigated this system with arachidonic acid and characterized the primary products. By the early stages of autoxidation (∼10% conversion of arachidonic acid), the major products detected by LC-MS and HPLC-UV were the 14,15-, 11,12-, and 8,9-EETs, with the 5,6-EET mainly represented as the 5-δ-lactone-6-hydroxyeicosatrienoate as established by 1H-NMR. The EETs were mainly the cis epoxides as expected, with minor trans configuration EETs among the products. 1H-NMR analysis in four deuterated solvents helped clarify the epoxide configurations. EET formation in monolayers involves intermolecular reaction with a fatty acid peroxyl radical, producing the EET and leaving an incipient and more reactive alkoxyl radical, which in turn gives rise to epoxy-hydro(pero)xides and other polar products. The monolayer alignment of fatty acid molecules resembles the arrangements of fatty acids in cell membranes and, under conditions of lipid peroxidation, this intermolecular mechanism might contribute to EET formation in biological membranes. more...

Abstract Introduction Comorbidity with metabolic diseases indicates that lipid metabolism plays a role in the etiology of Alzheimer's disease (AD). Comprehensive lipidomic analysis can provide new insights into the altered lipid metabolism in AD. Method In this study, a total 349 serum lipids were measured in 806 participants enrolled in the Alzheimer's Disease Neuroimaging Initiative Phase 1 cohort and analyzed using lipid‐set enrichment statistics, a data mining method to find coregulated lipid sets. Results We found that sets of blood lipids were associated with current AD biomarkers and with AD clinical symptoms. AD diagnosis was associated with 7 of 28 lipid sets of which four also correlated with cognitive decline, including polyunsaturated fatty acids. Cerebrospinal fluid amyloid beta (Aβ1‐42) correlated with glucosylceramides, lysophosphatidylcholines and unsaturated triacylglycerides; cerebrospinal fluid total tau and brain atrophy correlated with monounsaturated sphingomyelins and ceramides, in addition to EPA‐containing lipids. Discussion AD‐associated lipid sets indicated that lipid desaturation, elongation, and acyl chain remodeling processes are disturbed in AD subjects. Monounsaturated lipid metabolism was important in early stages of AD, whereas the polyunsaturated lipid metabolism was associated with later stages of AD. Our study provides several new hypotheses for studying the role of lipid metabolism in AD. more...

A proposed beneficial impact of highly unsaturated “fish oil” fatty acids is their conversion by lipoxygenase (LOX) enzymes to specialized proresolving lipid mediators, including 12/15-LOX products from EPA and DHA. The transformations of DHA include formation of docosatrienes, named for the distinctive conjugated triene of the double bonds. To further the understanding of biosynthetic pathways and mechanisms, herein we meld together biosynthesis and NMR characterization of the unstable leukotriene A (LTA)-related epoxide intermediates formed by recombinant human 15-LOX-1, along with identification of the stable enzymatic products, and extend the findings into the 12/15-LOX metabolism in resident murine peritoneal macrophages. Oxygenation of EPA by 15-LOX-1 converts the initial 15S-hydroperoxide to 14S,15S-trans-epoxy-5Z,8Z,10E,12E,17Z-EPA (appearing as its 8,15-diol hydrolysis products) and mixtures of dihydroperoxy fatty acids, while mainly the epoxide hydrolysis products are evident in the murine cells. DHA also undergoes transformations to epoxides and dihydroperoxides by 15-LOX-1, resulting in a mixture of 10,17-dihydro(pero)xy derivatives (docosatrienes) and minor 7S,17S- and 14,17S-dihydroperoxides. The 10,17S-dihydroxy hydrolysis products of the LTA-related epoxide intermediate dominate the product profile in mouse macrophages, whereas (neuro)protectin D1, the leukotriene B4-related derivative with trans,trans,cis conjugated triene, was undetectable. In this study, we emphasize the utility of UV spectral characteristics for product identification, being diagnostic of the different double bond configurations and hydroxy fatty acid functionality versus hydroperoxide. LC-MS is not definitive for configurational isomers. Secure identification is based on chromatographic retention times, comparison with authentic standards, and the highly distinctive UV spectra. more...

Acylglycerophosphate acyltransferase 4 (AGPAT4)/lysophosphatidic acid acyltransferase delta catalyzes the formation of phosphatidic acid (PA), a precursor of triacyl­glycerol (TAG). We investigated the effect of Agpat4 gene ablation on white adipose tissue (WAT) after finding consistent expression across depots. Epididymal WAT mass was 40% larger in male Agpat4−/− mice than wild-type littermates, but unchanged in perirenal, retroperitoneal, and inguinal WAT and subscapular brown adipose tissue. Metabolic changes were identified in epididymal WAT that were not evident in perirenal WAT, which was analyzed for comparison. The total epididymal TAG content doubled, increasing adipocyte cell size without changing markers of differentiation. Enzymes involved in de novo lipogenesis and complex lipid synthesis downstream of phosphatidic acid production were also unchanged. However, total epididymal TAG hydrolase activity was reduced, and there were significant decreases in total ATGL and reduced phosphorylation of hormone-sensitive lipase at the S563 and S660 PKA-activation sites. Analysis of Agpats 1, 2, 3, and 5, as well as Gpats 1, 2, 3, and 4, demonstrated compensatory upregulation in perirenal WAT that did not occur in epididymal WAT. Our findings therefore indicate depot-specific differences in the redundancy of Agpat4 and highlight the molecular and metabolic heterogeneity of individual visceral depots. more...

Triglyceride (TG) concentration is used as a marker of cardiometabolic risk. However, diurnal and possibly weekday variation exists in TG concentrations. The objective of this work was to investigate weekday variation in TG concentrations among 1.8 million blood samples drawn between 2008 and 2015 from patients in the Capital region of Denmark. Plasma TG was extracted from a central clinical laboratory information system. Weekday variation was investigated by means of linear mixed models. In addition to the profound diurnal variation, the TG concentration was 4.5% lower on Fridays compared with Mondays (P < 0.0001). The variation persisted after multiple adjustments for confounders and was consistent across all sensitivity analyses. Out-patients and in-patients, respectively, had 5.0% and 1.9% lower TG concentrations on Fridays compared with Mondays (both P < 0.0001). The highest weekday variations in TG concentrations were recorded for out-patients between the ages of 9 and 26 years, with up to 20% higher values on Mondays compared with Fridays (all P < 0.05). In conclusion, TG concentrations were highest after the weekend and gradually declined during the week. We suggest that unhealthy food intake and reduced physical activity during the weekend increase TG concentrations which track into the week. This weekday variation may carry implications for public health and future research practice. more...

In recent years the short-chain fatty acid, 4-phenylbutyrate (PB), has emerged as a promising drug for various clinical conditions. In fact, PB has been Food and Drug Administration-approved for urea cycle disorders since 1996. PB is more potent and less toxic than its metabolite, phenylacetate (PA), and is not just a pro-drug for PA, as was initially assumed. The metabolic pathway of PB, however, has remained unclear. Therefore, we set out to identify the enzymes involved in the β-oxidation of PB. We used cells deficient in specific steps of fatty acid β-oxidation and ultra-HPLC to measure which enzymes were able to convert PB or its downstream products. We show that the first step in PB oxidation is catalyzed solely by the enzyme, medium-chain acyl-CoA dehydrogenase. The second (hydration) step can be catalyzed by all three mitochondrial enoyl-CoA hydratase enzymes, i.e., short-chain enoyl-CoA hydratase, long-chain enoyl-CoA hydratase, and 3-methylglutaconyl-CoA hydratase. Enzymes involved in the third step include both short- and long-chain 3-hydroxyacyl-CoA dehydrogenase. The oxidation of PB is completed by only one enzyme, i.e., long-chain 3-ketoacyl-CoA thiolase. Taken together, the enzymatic characteristics of the PB degradative pathway may lead to better dose finding and limiting the toxicity of this drug. more...

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS)1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog, ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, SMS-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate the head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with Glu permitting SMS-catalyzed CPE production and Asp confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes. more...

Vitreoscilla filiformis is a Gram-negative bacterium isolated from spa waters and described for its beneficial effects on the skin. We characterized the detailed structure of its lipopolysaccharide (LPS) lipid A moiety, an active component of the bacterium that contributes to the observed skin activation properties. Two different batches differing in postculture cell recovery were tested. Chemical analyses and mass spectra, obtained before and after mild-alkali treatments, revealed that these lipids A share the common bisphosphorylated β-(1→6)-linked d-glucosamine disaccharide with hydroxydecanoic acid in an amide linkage. Short-chain FAs, hydroxydecanoic and dodecanoic acid, were found in a 2:1 ratio. The two lipid A structures differed by the relative amount of the hexa-acyl molecular species and phosphoethanolamine substitution of the phosphate groups. The two V. filiformis LPS batches induced variable interleukin-6 and TNF-α secretion by stimulated myelomonocytic THP-1 cells, without any difference in reactive oxygen species production or activation of caspase 3/7. Other different well-known highly purified LPS samples were characterized structurally and used as standards. The structural data obtained in this work explain the low inflammatory response observed for V. filiformis LPS and the previously demonstrated beneficial effects on the skin. more...

Close contacts between organelles, often called membrane contact sites (MCSs), are regions where lipids are exchanged between organelles. Here, we identify a novel mechanism by which cells promote phospholipid exchange at MCSs. Previous studies have shown that phosphatidylserine (PS) synthase activity is highly enriched in portions of the endoplasmic reticulum (ER) in contact with mitochondria. The objective of this study was to determine whether this enrichment promotes PS transport out of the ER. We found that PS transport to mitochondria was more efficient when PS synthase was fused to a protein in the ER at ER-mitochondria contacts than when it was fused to a protein in all portions of the ER. Inefficient PS transport to mitochondria was corrected by increasing tethering between these organelles. PS transport to endosomes was similarly enhanced by PS production in regions of the ER in contact with endosomes. Together, these findings indicate that PS production at MCSs promotes PS transport out of the ER and suggest that phospholipid production at MCSs may be a general mechanism of channeling lipids to specific cellular compartments. more...

α-Chlorofatty aldehydes are generated from myeloperoxidase-derived HOCl targeting plasmalogens, and are subsequently oxidized to α-chlorofatty acids (α-ClFAs). The catabolic pathway for α-ClFA is initiated by ω-oxidation. Here, we examine PPAR-α activation as a mechanism to increase α-ClFA catabolism. Pretreating both HepG2 cells and primary mouse hepatocytes with the PPAR-α agonist, pirinixic acid (Wy 14643), increased the production of α-chlorodicarboxylic acids (α-ClDCAs) in cells treated with exogenous α-ClFA. Additionally, α-ClDCA production in Wy 14643-pretreated wild-type mouse hepatocytes was accompanied by a reduction in cellular free α-ClFA. The dependence of PPAR-α-accelerated α-ClFA catabolism was further demonstrated by both impaired metabolism in mouse PPAR-α−/− hepatocytes and decreased clearance of plasma α-ClFA in PPAR-α−/− mice. Furthermore, Wy 14643 treatments decreased plasma 2-chlorohexadecanoic acid levels in wild-type mice. Additional studies showed that α-ClFA increases PPAR-α, PPAR-δ, and PPAR-γ activities, as well as mRNA expression of the PPAR-α target genes, CD36, CPT1a, Cyp4a10, and CIDEC. Collectively, these results indicate that PPAR-α accelerates important pathways for the clearance of α-ClFA, and α-ClFA may, in part, accelerate its catabolism by serving as a ligand for PPAR-α. more...

SM is a fundamental component of mammalian cell membranes that contributes to mechanical stability, signaling, and sorting. Its production involves the transfer of phosphocholine from phosphatidylcholine onto ceramide, a reaction catalyzed by SM synthase (SMS) 1 in the Golgi and SMS2 at the plasma membrane. Mammalian cells also synthesize trace amounts of the SM analog ceramide phosphoethanolamine (CPE), but the physiological relevance of CPE production is unclear. Previous work revealed that SMS2 is a bifunctional enzyme producing both SM and CPE, whereas a closely related enzyme, sphingomyelin synthase-related protein (SMSr)/SAMD8, acts as a monofunctional CPE synthase in the endoplasmatic reticulum. Using domain swapping and site-directed mutagenesis on enzymes expressed in defined lipid environments, we here identified structural determinants that mediate head group selectivity of SMS family members. Notably, a single residue adjacent to the catalytic histidine in the third exoplasmic loop profoundly influenced enzyme specificity, with glutamic acid permitting SMS-catalyzed CPE production and aspartic acid confining the enzyme to produce SM. An exchange of exoplasmic residues with SMSr proved sufficient to convert SMS1 into a bulk CPE synthase. This allowed us to establish mammalian cells that produce CPE rather than SM as the principal phosphosphingolipid and provide a model of the molecular interactions that impart catalytic specificity among SMS enzymes.This article has been withdrawn by the authors. See the withdrawal notice here: April 1 erratum jlr. M068692ERR. more...

Branched fatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids with anti-inflammatory and anti-diabetic properties. Despite the possible link between endogenous FAHFA levels and nuclear factor erythroid 2-related factor 2 (Nrf2), their possible function as antioxidants and the mechanisms involved in this are unknown. Here, we investigate FAHFAs’ plausible antioxidant potential with reference to their effect on the Nrf2 levels, oxidative stress, and lipid droplet oxidation in human hepatocytes (C3A). Six authentic FAHFAs were chemically synthesized and performed activity-based screening by reporter gene assay. Among them, eicosapentaenoic acid (EPA) esterified 12-hydroxy stearic acid (12-HSA) and 12-hydroxy oleic acid (12-HOA) FAHFAs showed less cytotoxicity compared to their free fatty acids and potent activators of Nrf2. To define their mode of action, relative levels of nuclear Nrf2 were determined, which found a higher amount of Nrf2 in nucleus of cells treated with 12-EPAHSA compared to the control. Furthermore, 12-EPAHSA increased the expression of Nrf2-dependent antioxidant enzyme genes (NQO1, GCLM, GCLC, SOD-1, and HO-1). Fluorescence imaging analysis of linoleic-acid-induced lipid droplets (LDs) in C3A cells treated with 12-EPAHSA revealed the strong inhibition of small-size LD oxidation. These results suggest that EPA-derived FAHFAs as a new class of lipids with less cytotoxicity, and strong Nrf2 activators with plausible antioxidant effects via the induction of cytoprotective proteins against oxidative stress, induced cellular damage. more...

The catabolism of intracellular triacylglycerols (TAGs) involves the activity of cytoplasmic and lysosomal enzymes. Cytoplasmic TAG hydrolysis, commonly termed lipolysis, is catalyzed by the sequential action of three major hydrolases, namely adipose triglyceride lipase, hormone-sensitive lipase, and monoacylglycerol lipase. All three enzymes interact with numerous protein binding partners that modulate their activity, cellular localization, or stability. Deficiencies of these auxiliary proteins can lead to derangements in neutral lipid metabolism and energy homeostasis. In this review, we summarize the composition and the dynamics of the complex lipolytic machinery we like to call “lipolysome”. more...

The article deals with the inventive activity of the Department of Lipid Biochemistry of the Palladin Institute of Biochemistry from 1980 until now. The researchers of the department headed by N.M.Hula have found lipids of unusual chemical structure in mouse neuroblastoma cells C1300 N18 such as N-acylethanolamine (NAE) as well as its precursor N-acylphosphatidylethanolamine (NAPE). From then on a long-term work on the study of functional roles of these lipids began. more...

Environments combining JP-8 jet fuel exposure with heightened ambient noise may accelerate hearing loss induced by noise. To reduce animal use and facilitate kinetic modeling of this military aviation fuel, tissue-specific parameters are required, including water, protein, and lipid content. However, tissues involved in hearing, including cochlea, brainstem, frontal, and temporal lobe, have not been characterized before. Therefore, water content was determined by lyophilization of rat auditory tissues and the protein of the freeze dried remainder was quantified using a bicinchoninic acid assay. Lipids were extracted from fresh-frozen rat auditory tissues and separated into neutral lipids, free fatty acids, neutral phospholipids, and acidic phospholipids using solid phase extraction. Phospholipid fractions were confirmed by 31 P nuclear magnetic resonance analysis showing distinct phospholipid profiles. Lipid content in reference tissues, such as kidney and adipose, confirmed literature values. For the first time, lipid content in the rat auditory pathway was determined showing that total lipid content was lowest in cochlea and highest in brainstem compared with frontal and temporal lobes. Auditory tissues displayed distinct lipid fraction profiles. The information on water, protein, and lipid composition is necessary to validate algorithms used in mathematical models and predict partitioning of chemicals of future interest into these tissues. This research may reduce the use of animals to measure partition coefficients for prospective physiological models. [ABSTRACT FROM AUTHOR] more...

The objective of this research was to quantify the internal fat deposits in Santa Inês sheep and to characterize the associated lipid profile. Forty-two male lambs, with a slaughter weight of 28.8 ± 2.33 kg and an average age of 7–8 months were used. Samples were collected from omentum, mesenteric, kidney and pelvic fat deposits. The fatty acids were separated by gas chromatography coupled with flame ionization detection. Chromatograms, with data on retention times and the percentages of the areas of fatty acid, were recorded using Peak software (ARI Instruments, USA). We observed that the omentum deposit had greater weight because it is the most active adipose tissue, i.e., the most sensitive to lipolysis because it covers the entire stomach in animals. Fourteen saturated fatty acids, ten monounsaturated fatty acids, and ten polyunsaturated fatty acids were identified. The fatty acids most commonly found in sheep were myristyl (C14:0), palmitic (C16:0), stearic (C18:0) and oleic (C18:1). The fatty acid profile of the fat deposits points to the possibility of using these fats in the formulation of the meat-derived product, an alternative that would add economic and nutritional value to these products. [ABSTRACT FROM AUTHOR] more...

Production of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in plant seed oils has been pursued to improve availability of these omega-3 fatty acids that provide important human health benefits. Canola ( Brassica napus), through the introduction of 10 enzymes, can convert oleic acid (OLA) into EPA and ultimately DHA through a pathway consisting of two elongation and five desaturation steps. Herein we present an assessment of the substrate specificity of the seven desaturases and three elongases that were introduced into canola by expressing individual proteins in yeast. In vivo feeding experiments were conducted with 14 potential fatty acid intermediates in an OLA to DHA pathway to determine the fatty acid substrate profiles for each enzyme. Membrane fractions were prepared from yeast expression strains and shown to contain active enzymes. The elongases, as expected, extended acyl-CoA substrates in the presence of malonyl-CoA. To distinguish between enzymes that desaturate CoA- and phosphatidylcholine-linked fatty acid substrates, we developed a novel in vitro method. We show that a delta-12 desaturase from Phytophthora sojae, an omega-3 desaturase from Phytophthora infestans and a delta-4 desaturase from Thraustochytrium sp., all prefer phosphatidylcholine-linked acyl substrates with comparatively low use of acyl-CoA substrates. To further validate our method, a delta-9 desaturase from Saccharomyces cerevisiae was confirmed to use acyl-CoA as substrate, but could not use phosphatidylcholine-linked substrates. The results and the assay methods presented herein will be useful in efforts to improve modeling of fatty acid metabolism and production of EPA and DHA in plants. [ABSTRACT FROM AUTHOR] more...

The human blood-brain barrier (BBB) is the restrictive barrier between the brain parenchyma and the circulating blood and is formed in part by microvessel endothelial cells. The brain contains significant amounts of arachidonic acid (ARA), and docosahexaenoic acid (DHA), which potentially give rise to the generation of bioactive oxylipins. Oxylipins are oxygenated fatty acid metabolites that are involved in an assortment of biological functions regulating neurological health and disease. Since it is not known which oxylipins are generated by human brain microvessel endothelial cells (HBMECs), they were incubated for up to 30 min in the absence or presence of 0.1-mM ARA, eicosapentaenoic acid (EPA) or DHA bound to albumin (1:1 molar ratio), and the oxylipins generated were examined using high performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS). Of 135 oxylipins screened in the media, 63 were present at >0.1 ng/mL at baseline, and 95 were present after incubation with fatty acid. Oxylipins were rapidly generated and reached maximum levels by 2-5 min. While ARA, EPA and DHA each stimulated the production of oxylipins derived from these fatty acids themselves, ARA also stimulated the production of oxylipins from endogenous 18- and 20-carbon fatty acids, including α-linolenic acid. Oxylipins generated by the lipoxygenase pathway predominated both in resting and stimulated states. Oxylipins formed via the cytochrome P450 pathway were formed primarily from DHA and EPA, but not ARA. These data indicate that HBMECs are capable of generating a plethora of bioactive lipids that have the potential to modulate BBB endothelial cell function. [ABSTRACT FROM AUTHOR] more...

The group of conjugated fatty acids known as conjugated linoleic acid (CLA) isomers have been extensively studied with regard to their bioactive potential in treating some of the most prominent human health malignancies. However, CLA isomers are not the only group of potentially bioactive conjugated fatty acids currently undergoing study. In this regard, isomers of conjugated α-linolenic acid, conjugated nonadecadienoic acid and conjugated eicosapentaenoic acid, to name but a few, have undergone experimental assessment. These studies have indicated many of these conjugated fatty acid isomers commonly possess anti-carcinogenic, anti-adipogenic, anti-inflammatory and immune modulating properties, a number of which will be discussed in this review. The mechanisms through which these bioactivities are mediated have not yet been fully elucidated. However, existing evidence indicates that these fatty acids may play a role in modulating the expression of several oncogenes, cell cycle regulators, and genes associated with energy metabolism. Despite such bioactive potential, interest in these conjugated fatty acids has remained low relative to the CLA isomers. This may be partly attributed to the relatively recent emergence of these fatty acids as bioactives, but also due to a lack of awareness regarding sources from which they can be produced. In this review, we will also highlight the common sources of these conjugated fatty acids, including plants, algae, microbes and chemosynthesis. [ABSTRACT FROM AUTHOR] more...

Fragile X syndrome (FXS) is the most common form of familial mental retardation and one of the leading known causes of autism. The mutation responsible for FXS is a large expansion of the CGG repeats in the promoter region of the FMR1 gene resulting in the transcriptional silencing of the gene in the pathophysiology of Fragile X syndrome was hypothesized. 23 male patients affected by Fragile X syndrome (full mutation in the FMR1 gene) and 24 controls were included in the study. The serum levels of HDL-C were lower in FXS patients ( p < 0.001). The serum levels triacylglycerols were higher in FXS patients ( p = 0.007) Further study involving larger samples are necessary to confirm the results and define the health implications for abnormal lipid levels in FXS patients. [ABSTRACT FROM AUTHOR] more...

Glucose and glutamine are essential energy metabolites for brain tumor growth and survival under both normoxic and hypoxic conditions. Both metabolites can contribute their carbons to lipid biosynthesis. We used uniformly labeled [C]-U- d-glucose and [C]-U- l-glutamine to examine the profile of de novo lipid biosynthesis in the VM-M3 murine glioblastoma cells. The major lipids synthesized included phosphatidylcholine (PtdCho), phosphatidylethanolamine (EtnGpl), phosphatidylinositol (PtdIns), phosphatidylserine (PtdSer), sphingomyelin (Cer PCho), bis(monoacylglycero)phosphate (BMP)/phosphatidic acid (PtdOH), cholesterol (C), cardiolipin (PtdGro), and gangliosides. Endogenous lipid synthesis, using either glucose or glutamine, was greater in media without fetal bovine serum (FBS) than in media containing 10 % FBS under normoxia. De novo lipid synthesis was greater using glucose carbons than glutamine carbons under normoxia. The reverse was observed for most lipids under hypoxia suggesting an attenuation of glucose entering the TCA cycle. Lactate was produced largely from glucose carbons with minimal lactate derived from glutamine under either normoxia or hypoxia. Accumulation of triacylglycerols (TAG), containing mostly saturated and mono-unsaturated fatty acids, was observed under hypoxia using carbons from either glucose or glutamine. The data show that the incorporation of labeled glucose and glutamine into most synthesized lipids was dependent on the type of growth environment, and that the VM-M3 glioblastoma cells could acquire lipids, especially cholesterol, from the external environment for growth and proliferation. [ABSTRACT FROM AUTHOR] more...

Bis(monoacylglycero)phosphate (BMP) is a structural isomer of phosphatidylglycerol (PtdGro) with an unusual sn-1:sn-1′ fatty acyl configuration and is found almost exclusively in late endosomes/lysosomes. BMP comprises only about 1-2 % of the total phospholipids in most mammalian cells, but accumulates in tissues of humans and animals with lysosomal storage disorders including the gangliosidoses. Total BMP content was significantly greater in cells of macrophage/microglial origin than in cells of macroglial origin. BMP composition was similar in tumorigenic/metastatic macrophages and non-tumorigenic macrophages/microglia. Finally, BMP fatty acid composition differed between cells grown in culture and obtained in vivo suggesting an influence from growth environment. [ABSTRACT FROM AUTHOR] more...

Main conclusion: Plants have lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities. The combined action of LPCT and GPCAT provides a novel route of PC re-synthesis after its deacylation. Abstract: Phosphatidylcholine (PC) is the major lipid in eukaryotic membranes and has a central role in overall plant lipid metabolism. It is also the site of production of polyunsaturated fatty acids in plants. The recently discovered acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activity in yeast provides a novel route of re-synthesising PC via lysophosphatidylcholine (LPC) after its deacylation. This route does not require the degradation of the glycerophosphocholine (GPC) into free choline, the activation of choline to CDP-choline, nor the utilization of CDP-choline by the CDP-choline:diacylglycerol cholinephosphotransferase. We show here that GPCAT activities also are present in membrane preparations from developing oil seeds of safflower and other species as well as in membrane preparations of roots and leaves of Arabidopsis, indicating that GPCAT activity plays a ubiquitous role in plant lipid metabolism. The last step in formation of GPC, the substrate for GPCAT, is the deacylation of LPC. Microsomal membranes of developing safflower seeds utilized LPC in LPC:LPC transacylation reactions (LPCT activities) creating PC and GPC. The results demonstrate that safflower membranes have LPCT and GPCAT activities that represent novel reactions for PC acyl editing. The physiological relevance of these reactions probably has to await identification of the enzymes catalysing these reactions. [ABSTRACT FROM AUTHOR] more...

The biosynthesis of very-long-chain polyunsaturated fatty acids involves an alternating process of fatty acid desaturation and elongation catalyzed by complex series of enzymes. ω3 desaturase plays an important role in converting ω6 fatty acids into ω3 fatty acids. Genes for this desaturase have been identified and characterized in a wide range of microorganisms, including cyanobacteria, yeasts, molds, and microalgae. Like all fatty acid desaturases, ω3 desaturase is structurally characterized by the presence of three highly conserved histidine-rich motifs; however, unlike some desaturases, it lacks a cytochrome b5-like domain. Understanding the structure, function, and evolution of ω3 desaturases, particularly their substrate specificities in the biosynthesis of very-long-chain polyunsaturated fatty acids, lays the foundation for potential production of various ω3 fatty acids in transgenic microorganisms. [ABSTRACT FROM AUTHOR] more...

The prevalence of hypertension in sub-Saharan Africa is increasing rapidly, and treatment remains challenging. Although the use of l-carnitine in treatment has received much attention, studies reporting on physiological l-carnitine levels in hypertensives are limited. Our aim was to determine physiological levels of l-carnitine and acylcarnitines in African and Caucasian men, and to investigate associations between ambulatory blood pressure (BP) and carnitine levels. Participants included 101 African and 101 Caucasian teachers. Ambulatory BP measurements were conducted, and l-carnitine and acylcarnitine levels determined. African men showed significantly higher systolic BP ( p < 0.001), diastolic BP ( p < 0.001) and l-carnitine levels ( p = 0.01). In both ethnic groups, partial regression analyses revealed a positive association between BP and l-carnitine, although in Caucasians it was with systolic ( r = 0.20, p = 0.045), and in Africans with diastolic BP ( r = 0.23, p = 0.023). After adjusting for confounders, an independent positive association between systolic ( R = 0.37, β = 0.12, p = 0.041) and diastolic BP ( R = 0.39, β = 0.14, p = 0.018) and l-carnitine and long-chain acylcarnitines ( R = 0.38, β = 0.17, p = 0.005 and R = 0.39, β = 0.15, p = 0.011) were found, independent of ethnicity. Physiological l-carnitine levels were not only higher in Africans than in Caucasians but also above the expected reference range. Despite promising results on l-carnitine (and its short-chain derivatives) in hypertension treatment regimens, our findings paradoxically show that elevated BP is significantly associated with higher physiological l-carnitine and long-chain acylcarnitine levels. [ABSTRACT FROM AUTHOR] more...

This short review takes into consideration the status of lipidomics as issued from almost a decade of development. Because of the huge number of molecular species analyzed, there is a trend in subdividing lipidomics according to subdomains, in particular relating to the function of molecules. It is also pointed out that lipid imaging without the use of exogenous probes will help making relationships between molecular structures and the topography of lipid assemblies, especially in cellular compartments. Finally, a fluxomics approach is proposed for lipid molecular species, both in terms of compartments and biochemical metabolism. The example of fluxolipidomics of essential fatty acids toward their enzyme-dependent oxygenated metabolites and further toward their degradation products is developed. [ABSTRACT FROM AUTHOR] more...